Immunosensors: scFv-linker design for surface immobilization

ABSTRACT

An apparatus and methods for binding an analyte of interest in a sample are provided. The apparatus comprises a substrate with an exposed surface with an compound, that is electrostatically charged or capable of forming hydrogen bonds, provided bound to the solid substrate. A recombinant single chain antibody (scFv) molecule specific for the analyte of interest, having one or more amino acids with charged or hydrogen-bond forming sidechains in a linker polypeptide portion, is bound to the layer on the solid substrate. When the analyte of interest is present in the sample the scFv binds the analyte to the solid substrate. The apparatus can be used with an immunoglobulin layer to detect Fc receptors, so as to detect microorganisms such as  Staphylococcus aureus  having protein A or protein G.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 10/861,617, filed Jun. 4, 2004, which claims priority to U.S.Provisional Application Ser. No. 60/476,123, filed Jun. 5, 2003.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This research was supported by grants from the National Institutes ofHealth (NIH 1R21EB000672-01, 4R33EB000672-02, 5P30 CA68485-07, 5P30ES00267-36). The U.S. government has certain rights to this invention

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates generally to single chain fragmentvariables (scFv) antibody molecules. Specifically, the present inventionrelates binding scFv antibody molecules on a substrate that can bind ananalyte of interest to the surface.

(2) Description of the Related Art

Antibodies and antibody-based reagents are used in immunotherapy andsolid phase based applications including biosensors, affinitychromatography, and immunoassays. In 1988, Phage display technology wasdeveloped that allows the presentation of large peptide and proteinlibraries on the surface of filamentous phage, which leads to theselection of antibodies and antibody fragments, with high affinity andspecificity to almost any target. The smallest such antibody fragment isthe Fv, which is obtained by association of the variable domains of theheavy chain (VH) and the light chain (VL) of the antibody. Without theaccompanying Fc region, Fvs can dissociate rapidly into their singledomains, VH and VL and results in a complete loss of the function of theFv. Protein engineering, recombinant DNA cloning and expressiontechniques allow the production of small Fvs, which have the domains ofthe heterodimers stabilized in various ways. For example, recombinantFvs may contain flexible inter- or intra-chain linkers, connector'speptides, or extra disulfide bonds, and they often fully retain thebinding specificity and affinity of the corresponding antibody. ManyscFvs retain the specificity and have similar affinity with the originalantibody or the monovalent Fab fragment. The advantages of thephage-displayed recombinant antibodies over the conventional polycolonalor monocolonal antibodies are quick generation time, cheap productioncost, and importantly, accessibility to the antibody DNA for furthergenetic manipulations. However, their long circulating half-life is adisadvantage for tumor imaging and therapy. Furthermore, without Fcportion, it cannot initiate immunoresponse. Typically, the scFvs wereconjugated with a drug to target tumor cells. However, combining scFvwith drugs may increase the chance of toxicity.

While the related art antibodies bound to solid substrates, there stillexists a need for an improved system that can immobilize scFv to thesensor substrate (such as Au) so that it can bind analytes of interestto a substrate.

SUMMARY OF THE INVENTION

The present invention provides an apparatus comprising: a substrate withan exposed surface; a compound provided as a layer, bound to the solidsubstrate; and a plurality of recombinant single chain antibodies(scFv's) specific for the target molecule and bound to the compound onthe solid substrate, each scFv comprising an antibody variable lightchain (V_(L)) polypeptide specific for the target molecule, an antibodyvariable heavy chain (V_(H)) polypeptide specific for the targetmolecule, and a linker polypeptide covalently linking the antibodyvariable light chain (V_(L)) polypeptide to the antibody variable heavychain (V_(H)) polypeptide, the linker polypeptide having an amino acidsequence comprising one or more amino acids with side chains that bindto the compound, wherein the scFv immobilized on the solid substrate iscapable of binding the target molecule, when provided to the apparatus.

In further embodiments of the apparatus, the recombinant single chainantibodies (scFv's) are specific for immunoglobulins as the targetmolecules, the apparatus further comprising a plurality of theimmunoglobulins bound to the scFv molecules so that Fc regions of theimmunoglobulins are exposed as a binding layer for Fc receptors. In someembodiments, the target molecules are analytes of interest in a sampleand the apparatus detects whether the analyte of interest is present inthe sample. In some embodiments of the apparatus, the compound and theamino acid sidechains form electrostatic interactions. In someembodiments, the compound and the amino acid sidechains form hydrogenbonds. In some of the embodiments, the amino acids with sidechains arearginine or tyrosine. In further embodiments, the compound iselectrostatically charged. In some embodiments, the compound is ananionic polyelectrolyte or 2-mercaptoethanol. In some of theseembodiments, the anionic polyelectrolyte is poly(sodium4-styrenesulfonate) (PSS) or 11-mercaptoundecanoic acid (MUA). Infurther embodiments, the substrate is gold. In some preferredembodiments, the apparatus is provided as a binding component of animmunosensor. In some of these embodiments, the immunosensor is a quartzcrystal microbalance (QCM) device or a surface plasmon resonance (SPR)device. In still further embodiments, the apparatus is provided as amicrotiter plate for an ELISA assay or as an affinity matrix forimmunopurification. In still further embodiments, the amino acids withsidechains are separated by one or more spacer amino acids. In furtherembodiments, the spacer amino acids are selected from the groupconsisting of glycine and serine. In some embodiments, the amino acidsequence of the linker polypeptide comprises a series of two or morearginine-glycine (RG) repeats. In still further embodiments, the aminoacid sequence of the linker polypeptide comprises a sequence selectedfrom the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, SEQ ID NO:11, and SEQ ID NO:12.

The present invention provides a method of detecting an analyte ofinterest in a sample comprising: providing the sample; providing animmunosensor device having a component apparatus for binding the analyteof interest in a sample comprising: a substrate with an exposed surface;a compound provided as a layer bound on the solid substrate; and aplurality of recombinant single chain antibodies (scFv's) specific forthe analyte of interest bound to the compound on the solid substrateeach scFv comprising an antibody variable light chain (V_(L))polypeptide specific for an analyte, an antibody variable heavy chain(V_(H)) polypeptide specific for the analyte, and a linker polypeptidecovalently linking the antibody variable light chain (VL) polypeptide tothe antibody variable heavy chain (VH) polypeptide, the linkerpolypeptide having an amino acid sequence comprising one or more aminoacids with sidechains that bind to the compound, wherein when theanalyte of interest is present in the sample the scFv binds the analyteto the solid substrate; applying the sample to the apparatus for a timesufficient for the scFv on the substrate to bind to the analyte ifpresent in the sample; and detecting the analyte bound to the scFv onthe solid substrate with the immunosensor device. In furtherembodiments, the immunosensor device is a quartz crystal microbalance(QCM) device or a surface plasmon resonance (SPR) device.

The present invention provides a method of binding an analyte ofinterest in a sample comprising: providing the sample; providing anapparatus for binding an analyte of interest in a sample comprising: asubstrate with an exposed surface; a compound provided as a layer boundon the solid substrate; and a plurality of recombinant single chainantibodies (scFv's) specific for the analyte of interest bound to thecompound on the solid substrate each scFv comprising an antibodyvariable light chain (V_(L)) polypeptide specific for an analyte, anantibody variable heavy chain (V_(H)) polypeptide specific for theanalyte, and a linker polypeptide covalently linking the antibodyvariable light chain (VL) polypeptide to the antibody variable heavychain (VH) polypeptide, the linker polypeptide having an amino acidsequence comprising one or more amino acids with sidechains that bind tothe compound, wherein when the analyte of interest is present in thesample the scFv binds the analyte to the solid substrate; and incubatingthe sample to the apparatus for a time sufficient for the scFv on thesolid substrate to bind to the analyte if present in the sample. Infurther embodiments, the apparatus is a microtiter plate for an ELISAassay or a microarray. In still further embodiments, the apparatus is anaffinity column.

The present invention provides a method of determining whether ananalyte with an Fc receptor is present in a sample comprising: providingthe sample; providing an immunosensor device having a component anapparatus comprising: a substrate with an exposed surface; a compoundprovided as a layer, bound to the solid substrate; a plurality ofrecombinant single chain antibodies (scFv's) specific for animmunoglobulin and bound to the compound on the solid substrate, eachscFv comprising an antibody variable light chain (V_(L)) polypeptidespecific for the immunoglobulin, an antibody variable heavy chain(V_(H)) polypeptide specific for the immunoglobulin, and a linkerpolypeptide covalently linking the antibody variable light chain (VL)polypeptide to the antibody variable heavy chain (VH) polypeptide, thelinker polypeptide having an amino acid sequence comprising one or moreamino acids with sidechains that bind to the compound, wherein the scFvis capable of binding the immunoglobulin to the solid substrate; and aplurality of the immunoglobulins bound to the scFv molecules so that Fcregions of the immunoglobulins are exposed as a binding layer for the Fcreceptor; applying the sample to the apparatus for a time sufficient forthe scFv on the substrate to bind to the Fc receptor if present in thesample; detecting the Fc receptors in the sample bound to the scFv onthe solid substrate with the immunosensor device; and determiningwhether the analyte is present in the sample by the result in step (d).

In further embodiments of the method, the immunosensor device is aquartz crystal microbalance (QCM) device or a surface plasmon resonance(SPR) device. In further embodiments, the Fc receptor is Protein A orProtein G. In still further embodiments, the analyte with the Fcreceptor is Staphylococcus aureus. In still further embodiments, theimmunoglobulin is IgG.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a scheme of an idealized representation of the differentself-assembled surfaces: MUA/scFv-RG3 Au surface or PSS/scFv-RG3 on agold (Au) QCM surface.

FIGS. 2A and B are schematic representations of the detection of ProteinA bacteria, such as S. aureus using a sandwich antibody assay foramplification. FIG. 2A shows the protein A on the surface of the S.aureus, which is bound to the Fc portion of IgG. FIG. 2B shows theanionic layer that binds the scFv-RG3 to the gold (Au) surface of theQCM.

FIG. 3A illustrates an idealized representation of scFv-RG3 with thelinker sequence RGRGRGRGRSRGGGS (SEQ ID NO:5). The linker sequences forscFv-RG3, scFv-ZnS4 and scFv-CdS6 are shown on the right. FIG. 3B showidealized representations of the different self-assembled surfaces:MUA/scFv-RG3 Au surface or PSS/scFv-RG3 or Au surface.

FIGS. 4A and B are graphs that show the characterization of A10BscFv-RG3 (A10B-RG3), scFv-cys (A10B-Cys), scFv-His (A10B-His), scFv-ZnS4(A10B-ZnS4), scFv-CDS6 (A10B-CDS6), scFv-F11, scFv-RS (A10B-RS)antibodies using HPR ELISA. FIG. 4A depicts ELISA results for A10BscFv-RG3, A10B-RS, scFv-Cys and scFv-His and negative control (scFv I-20specific for CYP1B1 P450, scFv D 11-Cys specific for isoketal proteinadduct, A10B-D2 monoclonal antibody) antibodies on (from left to rightin each set) rabbit IgG, human IgG, goat IgG, rat IgG, bovine IgG andbovine serum albumin (BSA). FIG. 4B depicts ELISA results for varyingconcentrations of rabbit IgG binding with A10B RG3.

FIG. 5A shows CVs of 1 mM K₄Fe(CN)₆/K₃Fe(CN)₆ in 0.1 M NaClO₄ on baregold electrode, MUA, MUA/RG3, and MUA/RG3 binding with rabbit IgGmodified electrodes. Scan rate, 50 mV/s. FIG. 5B shows EIS Nyquistplots. Frequency range is 0.1 Hz-100 kHz. Bias potential equals to opencircuit potential. AC amplitude is 10 mV.

FIG. 6A shows frequency change vs. time and FIG. 6B shows frequencychange vs. [rabbit IgG]₀ when various concentrations of rabbit IgG wereadded to the MUA/scFv-RG3 modified Au QCM electrodes. Finalconcentrations of rabbit IgG were from 0.22 nM to 132 nM (FIGS. 6A, B)in 1 ml PBS buffer. The solutions were stirred during all themeasurements.

FIG. 7 shows frequency change vs. time curves when (a) FBS, (b) goatanti rabbit IgG Fab, (c) anti human IgG, and (d) yeast; curve B: rabbitIgG were added to PSS/scFv-RG3 (Curve A) and MUA/scFv-RG3 (Curve B)modified Au QCM sensor cells in 1 ml PBS buffer.

FIG. 8 is a comparison of scFv based QCM sensors' sensitivity. A)MUA/scFv-RG3 B) PSS/scFv-RG3, C) scFv-cys, D) scFv-RG3, E) scFv-His, andF) scFv-biotin pre-coated surfaces. All modified QCM sensors wereexposed to 132 nM rabbit IgG.

FIG. 9 are frequency change vs. time curves when MUA/scFv-RG3,C₁₂H₂₅SH/scFv-RG3, and NH₅CS/scFv-RG3 modified QCM sensors were exposedto the 132 nM rabbit IgG.

FIG. 10 are frequency change vs. time curves when different modified QCMsensors were exposed to the 132 nM rabbit IgG. A) MUA/scFv-RG3, B)PSS/scFv-RG3, C) MUA/scFv-ZnS4, D) PSS/scFv-ZnS4, E) MUA/scFv-CdS6, andF) PSS/scFv-CdS6 modified QCM sensors.

FIG. 11 is a plot of [rabbit-IgG]₀/ΔM vs. [rabbit-IgG]₀ for MUA/scFv-RG3modified QCM sensor.

FIG. 12 is a representation of a whole antibody and a scFv, with theantibody heavy-chain (V_(H)) and light-chain (V_(L)) variable domainsthat are connected by a peptide linker to stabilize the molecule.

FIG. 13 shows the system without template (left) and with template(right), illustrating how the template helps the scFv to be immobilizedwith desired orientation.

FIG. 14 shows frequency change vs. time curves when protein A was addedto (a) MUA/scFv-RG3 coupling with rabbit IgG surface (MUA/scFv-RG3/IgGsurface); (b) randomly orientated rabbit IgG surface; (c) rabbit IgGcoupling onto the protein A surface; (d) MUA/scFv-RG3 surface withoutrabbit IgG (negative control).

FIG. 15 shows frequency vs. time curves. MUA/scFv-RG3/IgG surface wasexposed to 2.6×10⁻⁸ cells/ml E. coli (negative control,), followed bythe addition of Staphylococcus aureus (2.5×10⁻⁸ cells/ml, 1.2×10⁻⁹cells/ml).

FIG. 16 shows the frequency vs, time curve when different concentrationsof Staphylococcus aureus were added to MUA/scFv-RG3 coupling with rabbitIgG surface.

FIG. 17 shows the frequency vs, time curve when MUA/scFv-RG3 couplingwith rabbit IgG surface was exposed to 2.6×10⁻⁸ cells/ml E. coli(negative control) and Staphylococcus aureus.

FIG. 18 shows the comparison of sensor specificity. 1.4×10⁹ cells/mlacid treated Staphylococcus aureus was added to rabbit IgG treatedMUA/scFv-RG3 sensor (Bar A); 1.4×10⁹ cells/ml non-treated Staphylococcusaureus was added to rabbit IgG treated MUA/scFv-RG3 sensor (Bar B);2.6×10⁸ cells/ml E. coli was added to rabbit IgG treated MUA/scFv-RG3sensor (Bar C). (D-F control surfaces) 1.4×10⁹ cells/ml acid treatedStaphylococcus aureus were added to random orientated rabbit IgG surface(D); MUA/scFv-RG3 surface (without rabbit IgG) surface (E); and mannosesurface (F).

FIG. 19 shows the SPR response difference between A10B scFv-Cysbiosensor reference cell (FC1) and sample cell (FC2). The graph containsseven injection steps that correspond to seven different concentrationsof rabbit IgG, 6.25, 12.5, 25, 50, 100, and twice 200 μg/mL or 0.042,0.083, 0.17, 0.33, 0.67, and twice 1.3 μM in that order. In thisexperiment, FC1 has the injection order of A10B-YG, R-IgG. FC2 has theinjection order of 2-mercaptoethanol, A10B-YG, R-IgG.

FIG. 20 shows ELISA results of sensitivity and specificity of A10B YGand A10B RG3.

FIG. 21 shows in this experiment (1) FC1 has the injection order ofA10B-YG, R-IgG. FC2 has the injection order of 2-mercaptoethanol,A10B-YG, R-IgG. (2) 30 μg/mL 0.3 M Zwittergent 3-08 pH 2.0. (3) repeatthe experiment of (1)

DETAILED DESCRIPTION OF THE INVENTION

All patents, patent applications, government publications, governmentregulations, and literature references cited in this specification arehereby incorporated herein by reference in their entirety. In case ofconflict, the present description, including definitions, will control.

The term “bacteria” as used herein refers to include both gram-positiveand gram-negative bacteria.

The term “immunosensor” as used herein refers to any immunosensingapparatus such as, but not limited to, quartz crystal microbalance (QCM)devices, surface plasmon resonance (SPR) devices, protein microarrays(ie. antibody microarrays), microbeads, and protein sensor chips.

The term “microorganism” as used herein refers to any microorganism,including but not limited to, bacteria and fungi.

The term “QCM” as used herein refers to a quartz crystal microbalance.Any quartz crystal microbalance devices can be used in the presentinvention including, but not limited to QCM devices available fromMaxtek Inc. of Santa Fe Springs, Calif. Other QCM devices which can beused in the present invention are described in U.S. Pat. No. 4,236,893to Rice, U.S. Pat. No. 4,242,096 to Oliveira et al., U.S. Pat. No.4,246,344 to Silver III, U.S. Pat. No. 4,314,821 to Rice, U.S. Pat. No.4,735,906 to Bastiaans, U.S. Pat. No. 5,314,830 to Anderson et al., U.S.Pat. No. 5,932,953 to Drees et al., and U.S. Pat. No. 6,087,187 toWiegland et al., U.S. Pat. No. 6,890,486 to Penelle, U.S. Pat. No.6,848,299 to Paul et al., U.S. Pat. No. 6,706,977 to Cain et al., U.S.Pat. No. 6,647,764 to Paul et al., U.S. Pat. No. 6,492,601 to Cain etal., U.S. Pat. No. 6,439,765 to Smith, U.S. Pat. No. 6,190,035 to Smith,U.S. Pat. No. 6,106,149 to Smith, U.S. Pat. No. 5,885,402 to Esquibel,U.S. Pat. No. 5,795,993 to Pfeifer et al., U.S. Pat. No. 5,706,840 toSchneider, U.S. Pat. No. 5,616,827 to Simmermon et al., U.S. Pat. No.5,484,626 to Storjohann et al., U.S. Pat. No. 5,282,925 to Jeng et al.,U.S. Pat. No. 5,233,261 to Wajid, U.S. Pat. No. 5,201,215 to Granstaffet al., U.S. Pat. No. 4,999,284 to Ward et al., and U.S. Pat. No.4,788,466 to Paul et al. Examples of control circuitry for quartzcrystal microbalances and methods for detecting materials usingpiezoelectric resonators are described in U.S. Pat. No. 5,117,192 toHurd and U.S. Pat. No. 5,932,953 to Drees et al. Each of the abovereferences are hereby incorporated herein by reference in theirentirety.

The term “surface” as used herein refers to any solid surface. In someembodiments, the solid surfaces are QCM electrode surfaces.

The term “SPR” as used herein refers to a surface plasmon resonance. AnySPR device can be used in the present invention including, but notlimited to, a Biocore system (GE Healthcare) or the SPR biosensor deviceas described in U.S. patent application Ser. No. 11/581,260 to Xiao andZeng.

U.S. patent application Ser. No. 10/861,617 to Zeng et al., herebyincorporated herein by reference in its entirety, discloses an improvedpiezoimmunosensor. Zeng et al. teach of the single chain fragmentvariable (scFv)'s capability and potential as a superior new type ofimmuno-recognition element by protein engineering the metal bindingamino acids (i.e. cysteine, histidine and biotin) into the peptidelinker which links the heavy (V_(H)) and the light (V_(L)) chainvariable domains. This allows the direct immobilization of scFv in theirnative state on the gold surface and the antigen-binding site isoriented toward the solution phase. Three papers have been published inyear 2005, 2006 and 2007 on this topic. Twelve A10B scFv constructs weremade as shown in Table 1. Six scFv that is biotinylated were also madefor binding with CYp1b1 enzyme. In these embodiments, the recombinantsingle chain antibody (scFv) molecule utilized in the present inventioncan comprise a first variable chain polypeptide having a first aminoacid sequence with an scFv amino terminus and a carboxy terminus, whichis an antibody variable light chain (V_(L)) or an antibody variableheavy chain (V_(H)) polypeptide specific for an analyte of interest; asecond variable chain polypeptide having a second amino acid sequencewith an amino terminus and an scFv carboxy terminus, which is anantibody variable light chain (V_(L)) or an antibody variable heavychain (V_(H)) polypeptide specific for the analyte of interest; and alinker polypeptide having a third amino acid sequence comprising one ormore amino acids having charged sidechains covalently linking thecarboxy terminus of the first variable chain polypeptide to the aminoterminus of the second variable chain polypeptide. In some embodimentsof the present invention, the recombinant single chain antibody (scFv)molecule further comprises a tagged amino acid sequence at the scFvcarboxy terminus. In further embodiments, the recombinant single chainantibody (scFv) molecule further comprises a tagged amino acid sequenceat the scFv amino terminus. In further embodiments, the scFv comprises abiotin tag.

We have recently realized the scFv's capability and potential as ansuperior new type of immuno-recognition elements by protein engineeringthe metal binding amino acids (i.e. cysteine and histidine) into thepeptide linker that links the heavy (V_(H)) and the light (V_(L)) chainvariable domains. This allows the direct immobilization of scFv in theirnative state on the gold surface and the antigen-binding site isoriented toward the solution phase. Our studies show that the specificorientation of scFv antibodies consistently increases theanalyte-binding capacity of the surfaces, with up to three-foldimprovements over surfaces with randomly oriented monoclonal antibody,five-fold improvements over surfaces with randomly oriented antibody Fabfragments. Literature shows that in many scFvs there seems to be littleeffect of these linker variations on affinity or stability of the Fv.However, the linker sequence can affect the yield of functional Fvs thatare obtained from refolding of inclusion antibodies. For example, it wasfound that when cysteines and histidines are incorporated into somerecombinant scFv antibodies or linkers, scFv antigen-binding activitycan be disrupted through inter-scFv disulfide bond formation and scFvaggregation; or scFv bacterial protein expression can be substantiallyreduced (J. Immun. Methods, 242 (2000)101-114).

We also demonstrated scFv with biotin tag for detection of P450 CYP1B1enzyme, an cancer biomarker (shown in inventor Zeng's publication“Recombinant Antibody Pizeoimmunosensors for the detection of CytochromeP450 1B1”, Analytical Chemistry Vol. 79, No. 4, pp. 1283-1289 (2007).

We also demonstrated that the scFv biosensor system can be used toprovide a platform immobilization strategy to build rigid IgG Fcreceptor layers. Just as importantly, the ability of immunoglobulins toreact with other molecules at sites located outside theantigen-combining site, related to the effector functions of antibodies,are the most important part of the immune response. These include fromsuch well-known reactions as the activation of the complement cascadeand the activation or the inhibition of cells after binding with the Fcreceptors to transportation of immunoglobulins through cell membranes.As shown in FIG. 1, monomeric scFv allows uniform 2:1 binding withrabbit IgG CH1 region, that results in a highly oriented IgG Fc portionpointing toward solution phase for the detection of Fc receptors. Thisarrangement was proven to be feasible by the detection of protein A, awell used Fc receptor in staphylococcal bacteria cell lysate. Detectionof protein A in bacteria lysate is challenging due to the presence ofexcess antibody in the analyte sample that seriously suppresses theresponse of an assay by competing for the Fc binding sites on protein A,thereby reducing the sensitivity of the assay. We have demonstrated thatthe specific orientation of Fc capture agents consistently increases theanalyte-binding capacity of the surfaces, with up to a seven-foldimprovement over surfaces with randomly oriented capture agents.Randomly attached IgG could not be packed at such a high density and hada lower specific activity. These results emphasize the importance of theimmobilization of capture reagents to surfaces such that their bindingsites are oriented towards the solution phase.

TABLE 1 A10B scFv constructs A1013-RS GGGGSGGGGSGGGGS SEQ ID NO: 9A10B-cys CGGGSGGGGSGGGGS SEQ ID NO: 8 A10B C4 SHGGHGGGGSGGGGS SEQ ID NO:13 A10B C-11 SHGGHGGGGSGGGGS linker: [has His-tagged (HHHHH; SEQ ID NO:13 H = histidine) sequence His-tag: on the C-terminus of SEQ ID NO: 14the scFv)] A10BMG4 MGGMSGGGGSGGGGS SEQ ID NO: 15 A10B YG YGGYSGGGGSGGGGSSEQ ID NO: 11 A10B WG WGGYSGGGGSGGGGS SEQ ID NO: 12 A10B FP1SVSVGMKPSPRP SEQ ID NO: 4 A10B ZnS4 VISNHAGSSRRL SEQ ID NO: 2 A10B Cds6PWIPTPRPTFTG SEQ ID NO: 3 A10B RG3 RGRGRGRGRGR SEQ ID NO: 1 A10B Mouseand rat antibodies contain two Biotinylated naturally occurring lysineswhich are located near the C-terminus of every antibody light chainvariable region. The, free amines located on these two lysines, on otherlysines that may be present at other locations in an scFv, or at thescFv amino terminus, can be readily biotinylated using commerciallyavailable biotinylation reagents, without destroying the scFvantigen-binding activity or specificity.

The present invention applies generally to any technique that utilizesrigid films that are built up for non-label detection, such as QCM orSPR of various reagents from proteins to cells or bacteria. The peptidechain length, amino acid composition, specific sequence, net charge atneutral pH and hydrophobicity, are features that can be used to designan scFv which can be directly immobilized on the gold surface ortemplate surface. For example, 15-mer unmodified peptides, there areabout 1024 diverse sequences when considering the twenty common aminoacids alone. Thus, the linker peptide design provides huge possibilitiesfor surface coupling methodology. This feature, in conjunction with thereal-time, non-labeled characters of QCM and/or SPR, presents a widelyapplicable protein immobilization technology for investigating theprotein-protein interactions in general. This illustrates that QCM canbe an indispensable technique in mapping the ligand-receptorinteractions that is affordable to most investigators in life scienceresearch. The scFv of the present invention can be applied tobiosensors, immunosensors, ELISA.

In one embodiment, monomeric single chain fragment variable (scFv)molecules allow uniform 2:1 binding with rabbit IgG CH1 region, thisresults a highly oriented IgG Fc portion pointing toward the solutionphase for the detection of Fc receptors. FIGS. 2A and B are schematicrepresentations of the detection of Protein A bacteria, such as S.aureus using a sandwich antibody assay for amplification. As illustratedin FIGS. 2A and B, by employing this concept we can detectStaphylococcus aureus bacteria with great amplification by binding therabbit IgG Fc portion to protein A, a membrane protein of Staphylococcusaureus bacteria, which is a gram positive bacteria. FIG. 2A shows theprotein A on the surface of the S. aureus, which is bound to the Fcportion of IgG. FIG. 2B shows the anionic layer that binds the scFv-RG3to the gold (Au) surface of the QCM.

We have demonstrated that the disadvantages of recombinant antibodyobserved in immunotherapy work as the benefits for immunosensing i.e.protein microarrays, microbeads, and protein sensor chips. Forimmunosensing, the intensity of specific signal produced on animmunosensor is related to the amount of analyte that is captured fromthe biological mixture by the immobilized antibody (ie. the “captureagent”). This in turn is a function of the surface density andfractional activity of the capture agents. The Fc portion is not neededand can detrimentally serve as a non-specific adsorption site. Thesmaller size of scFvs, as compared to commonly used monoclonalantibodies, increased the surface density and reduces the non-specificadsorption that greatly improved the sensor sensitivity and specificity.

The present invention provides methods for the immobilization of scFvsthat does not require the incorporation of cysteine or histidineresidues as metal binding amino acids. The immobilization methods takeadvantage of the limitless flexibility of antibody engineering for thescFvs with the inherent quick, clean, high fidelity characters ofsurface coupling chemistry (e.g. electrostatic, hydrogen bonding, orcovalent attachment) to attach scFvs to pre-formed functionalizedself-assembled monolayers. In one embodiment of the present invention,six arginines, which were separated by glycine (G) or serine (S) asspacers, were incorporated in the linker to form a 15-mer peptide linkerhaving the sequence RGRGRGRGRSRGGGS (SEQ ID NO:5). Since argininecontains positive charged side chain at neutral pH and can beimmobilized on the surface by electrostatic interaction with thenegative charged template. The polycationic peptide was engineered intothe A10B scFv model system. In some embodiments of the presentinvention, two functional template surfaces, poly(sodium4-sterenesulfonate) (PSS), and 11-mercaptoundecanoic acid (MUA) wereused for the specific adsorption of hexa-arginine linked scFv(scFv-RG3). Our results show that the hexa-arginine linked scFv(scFv-RG3) were efficiently adsorbed on the negative charged functionalsurface via an electrostatic interaction. Different immobilizationmethods based on the specific peptide linked scFvs were examined,including scFv-GR3/MUA, scFv-RG3/PSS, scFv-RG3/bare-gold,scFv-cys/bare-gold, scFv-His/bare-gold, and scFv-biotin/Avidin methods.The test showed that the scFv-RG3 and MUA coupling method provided thebest sensitivity and selectivity.

The location and the generality of above strategies were further studiedby engineering addition A10B scFv with positive charged aminoacids intolinker peptides. A10B ZnS4 scFv with linker sequence of VISNHAGSSRRL(SEQ ID NO:2), A10B scFv CdS6 with linker sequence of PWIPTPRPTFTG (SEQID NO:3), A10B scFv FP1 with linker sequence of SVSVGMKPSPRP (SEQ IDNO:4) were made.

The methods we demonstrated herein can be used for building up rigidfilms for acoustic detection of various biological antigens fromproteins to cell or bacterial. In contrast to SPR technique that has alimit of the thickness of the biofilms, i.e. less than 200 nm, QCMsensor detects only those materials that are acoustically coupled to thesensor surface and theoretically it has no limits regarding thethickness of the film. However, it requires the immobilized biofilmswith high rigidity so it can acoustically couple with quartzoscillation. The peptide chain length, amino acid composition, specificsequence, net charge at neutral pH, and hydrophobicity can each becontrolled and have dramatic influence on the performance of scFv basedQCM sensors. For example, for 15-mer unmodified peptides there are about1024 diverse sequences when considering the twenty common amino acidsalone. Thus, the linker peptide design provides a huge number ofpossibilities for a surface coupling methodology. This aspect of thepresent invention, in conjunction with the real-time, non-labeledcharacter of QCM, presents a widely applicable protein immobilizationtechnology. This can be used for investigating protein-proteininteractions in general and promises that QCM will be an indispensabletechnique in mapping the ligand-receptor interactions, while beingaffordable to most investigators in life science research.

EXAMPLE 1

In this example, an A10B scFv RG linker with positively charged RGlinkers were successfully used to couple to a negatively charged goldsurface. This coupling method has shown the best performance whencompared with other linker designs including cysteine and histidine.This approach has broad applicability since a negatively charged linker,such as a linker with arginine amino acid residues, could be coupled topositively charged gold template. The scFv immobilization was based onpre-formed self assemble monolayer (SAM) templates incorporated withvarious properties. These properties, such as negative charge, positivecharge, and hydrogen bonding, can further anchor the scFvs with a linkerthat is designed to match those interactions on the SAM template to formorientated scFv layers. The immobilized scFvs can be used for proteinrecognition for clinical and environmental applications, and also can beused to detect bacteria.

Chemicals and materials: Rabbit IgG (cat# 1-5006), bovine serum albumin(BSA, cat# A-4503), goat anti-rabbit IgG (cat# R-2004), and goatanti-human IgG (cat# I-3382) were purchased from Sigma Inc. Goatanti-rabbit IgG Fab fragment (cat# 111-007003) was purchased fromJackson Immunolabs. Peptides RGRGRGRGRSRGGGS (SEQ ID NO:5) and RGRG (SEQID NO:6) were purchased from Openbiosystems Inc. The peroxidaseconjugated Anti-E tag monoclonal antibody (cat# 27941301) was obtainedfrom Amersham. Poly(sodium 4-sterenesulfonate) (PSS, cat# 527483,MW:70,000), 11-mercaptoundecanoic acid (MUA, cat# 450561),1-Dodecanethiol, (cat# 471364) were purchased from Sigma-Aldrich.Phosphate buffered saline (PBS), pH 7.2 (Gibco BRL #20012-027), fetalbovine serum (FBS) (Gibco BRL #16000-044). All other chemicals (Aldrich)are reagent grade and used as received.

Preparation and purification of Anti Rabbit IgG ScFv, clone A10B RG3:Modified anti rabbit IgG scFv from clone A10B RG3 with theRGRGRGRGRSRGGGS linker (SEQ ID NO:5) was tested by ELISA. Data confirmedthese scFv is highly specific, efficient and effective against rabbitIgG. The bacterial clone producing A10B RG3 scFv reacted with rabbit IgGwas inoculated and cultured in 500 ml of 2×YT+AG medium which containsBacto Tryptone 17 g, Bacto Yeast extract 10 g, sodium chloride 5.0 gmAmpicillin 100 mg and Glucose 20 g per liter. E. coli bacteria wereincubated at 30° C. overnight with shaking at 125 rpm and thencentrifuged down to pellet bacterial cells. The cell pellet was thenre-suspended in 500 ml of 2×YT+AI (Ampicillin and IPTG 1 mM) medium andthen incubated at 30° C. with shaking at 125 rpm overnight. Theperiplasmic extract was made by incubating bacterial pellet in 25 ml of1×TES and 46 ml of ⅕ TES. Well re-suspended suspension was then placedon ice for at least one hour by vigorous shaking. The cell lysate wascentrifuged at 5,000 rpm. Anti-E tag affinity columns used for purifyingthe supernatants containing soluble E-tagged scFv. The concentration ofpurified scFv was tested by spectrophotometer at OD₂₈₀ nm. Thespecificity and binding activity of this purified scFv antibody torabbit IgG was analyzed by Enzyme-Linked Immunosorbent Assay (ELISA).

Recombinant scFv characterization by ELISA: ELISA was used tocharacterize the soluble recombinant scFv antibody A10B-RG3. Wells of a384 well microtiter plate (Nunc Cat. # 242757) were coated with serialdiluted rabbit IgG. Diluted soluble scFv antibody A10B-RG3 withRGRGRGRGRSRGGGS (SEQ ID NO:5) linker at the concentration of 2 μg/ml wasapplied to wells of a microtiter plate that was pre-coated with RabbitIgG. Since the A10B scFv has an E-tagged stemming from the rodentlibrary, the scFv bound to antigen was detected with the horseradishperoxidase (HRP)-conjugated anti-E tag monoclonal antibody (GEHealthcare), i.e. hydrogen peroxide and ABTS as a color indicator wasthen finally added to wells. A microtiter plate reader, operating at 405nm, was used to determine the absorbance readings for each well in anELISA and the results of the antigen-binding activity was analyzed.

A10B ScFv immobilization: The non-polished gold quartz surface wascleaned sequentially with mixed concentrated acid solution (H₂SO₄/HNO₃in 1:1 v/v), biograde water, and ethanol three times. Then, the surfacewas rinsed with biograde water and dried with nitrogen. The freshlywashed gold surface was first immersed either into 4 mM MUA solutionovernight or in 2 mg/ml PSS solution for 1-2 hours to form self-assemblyanionic charged layer. The anionic charged gold surface was thenimmersed into A10B scFv-RG3 solution for 8 hours, followed by thetreatment of blocking reagent, 0.1% BSA, for 0.5 hour. After theincubation, the excess scFvs on the surface of QCM was washed away withPBS buffer and biograde water. The MUA/scFv-RG3 or PSS/scFv-RG3 modifiedsurface was dried under nitrogen, and used for the detection. A similarprocedure was applied to form the PSS/scFv-RG3 modified surface.

QCM measurements: The gold quartz crystal electrode used through outthis example was AT cut quartz that coated with 1000 Å gold in anapproximate 0.23 cm² geometric area (International Crystal Company,Oklahoma). It was cleaned as above and modified with scFvs. Then it wasmounted in a Kel-F cell sealed by two O-rings and filled with 1 mL PBSbuffer. The cell was continuously stirred during the measurement and wasplaced in a Faraday cage to reduce the potential electromagneticalnoise. The frequency change and the damping resistance change caused bythe analyte addition were monitored by the Network/Spectrum/ImpedanceAnalyzer (Agilent 4395A).

Electrochemical Characterization: The gold QCM electrode was used as theworking electrode. A platinum wire and a saturated calomel electrode(SCE) were used as counter and reference electrodes respectively. Cyclicvoltammetry (CV) and electrochemical impedance spectroscopy (EIS) werecarried out in a solution of 0.1 M NaClO₄ containing 1 mMK₃Fe(CN)₆/K₄Fe(CN)₆ and performed using a Parstat 2263 advancedelectrochemical system (Princeton Applied Research).

Results and Discussion.

HRP ELISA quantification and characterization of A10B scFvs: AllA10B-scFv have an E-tag incorporated into the amino terminus of theprotein (E-tag: GAPVPYPDPLEPR, SEQ ID NO:7) stemming from the rodentlibrary. An E-tag is a specific linear epitope recognized bycommercially available HRP-conjugated anti-E-tag antibody, thusproviding a means for a HRP ELISA assay to characterize the affinity andspecificity of the binding of A10B-scFvs with rabbit IgG. FIG. 3Aillustrates an idealized representation of scFv-RG3 with the linkersequence RGRGRGRGRSRGGGS (SEQ ID NO:5). The linker sequences forscFv-RG3, scFv-ZnS4 and scFv-CdS6 are shown on the right. FIG. 3B showidealized representations of the different self-assembled surfaces:MUA/scFv-RG3 Au surface or PSS/scFv-RG3 or Au surface. The chemicalformulas for MUA and PSS for the anionic layer are shown in the upperright.

FIGS. 4A, and B illustrate the characterization of A10B scFv RG3,scFv-cys, scFv-his, scFv-ZnS4, scFv-CDS6, scFv-F11, scFv-RS antibodiesusing HPR ELISA. FIG. 4A depicts ELISA results for A10B scFv RG3, A10BRS, scFv-cys and scFv-his and negative control (scFv I-20 specific forCYP1B1 P450, scFv D 11-cys specific for isoketal protein adduct, A10B D2monoclonal antibody) antibodies on (from left to right) rabbit IgG,human IgG, goat IgG, rat IgG, bovine IgG and bovine serum albumin (BSA).FIG. 4A shows that anti E-tag affinity purified A10B scFv antibodies(i.e. A10B scFv-RG3, scFv-Cys, scFv-His, scFv-ZnS4, scFv-CDS6, scFv-F11,and scFv-RS) specifically bind to antigen rabbit IgG. A10B scFv-RG3,scFv-Cys, scFv-His, scFv-CDS6, and scFv-RS have similar affinity torabbit IgG. A10B scFv-ZnS4 and A10B scFv-F11 have relative smalleraffinity to rabbit IgG. Additionally, little non-specific interactionwith other antigens (i.e. IgGs of human, goat, rat and bovine) wereobserved by all A10B scFvs studied. This ELISA result shows that themodification of A10B scFv linker has little effect on A10B scFv antigenbinding affinity and specificity to rabbit IgG antigen. FIG. 4B depictsELISA results for varying concentrations of rabbit IgG binding with A10BRG3. FIG. 4B shows that the binding activity of A10B scFv-RG3 to rabbitIgG is dose-dependent and as low as 20 ng of rabbit IgG antigen can bedetected by A10B scFv-RG3.

Electrochemical Characterization of the MUA/scFv-RG3 Biosensor: Theintegrity of MUA/scFv-RG3 SAM and detection of rabbit IgG was probed bycyclic voltammetry and electrochemical impedance. K₄Fe(CN)₆/K₃Fe(CN)₆solution was used as electrochemical probe to test the surfaceintegrity. FIG. 5A shows CVs of 1 mM K₄Fe(CN)₆/K₃Fe(CN)₆ in 0.1 M NaClO₄on bare gold electrode, MUA, MUA/RG3, and MUA/RG3 binding with rabbitIgG modified electrodes. Scan rate, 50 mV/s. As shown in FIG. 5A, CV onthe bare gold surface gave reversible redox peaks. The Faradaic currentwas dramatically decreased once a SAM of MUA was formed on the gold (Au)surface and further attenuated when scFv-RG3 was coupled onto the MUASAM and bound with rabbit IgG. The electrochemical impedancespectroscopy was also used to text the surface passivation to electrontransfer. FIG. 5B shows EIS Nyquist plots. Frequency range is 0.1 Hz-100kHz. Bias potential equals to open circuit potential. AC amplitude is 10mV. As shown in Nyquist plots (FIG. 5B), while the electron-transferresistance (R_(et)) of the Fe(CN)₆ ^(IV)/Fe(CN)₆ ^(III) redox reactionsincreased drastically after the formation of the MUA SAM, and furtherincreased when scFv-RG3 was adsorbed onto MUA surface, and bound withrabbit IgG. These experiments supported the existence of highly packedMUA/scFv-RG3 layers and the binding of surface scFv to its correspondingantigen.

A10B scFv RG3 QCM sensor sensitivity to rabbit IgG: The A10B scFv RG3specific binds the CH1 region of rabbit IgG. HRP ELISA results aboveshow it has excellent sensitivity and specificity to rabbit IgG. As aresult, the A10B scFv RG3 QCM sensors immobilized either through MUA orPSS template was used to detect rabbit IgG with varying concentrations.FIG. 6A shows frequency change vs. time and FIG. 6B shows frequencychange vs. [rabbit IgG]₀ when various concentrations of rabbit IgG wereadded to the MUA/scFv-RG3 modified Au QCM electrodes. Finalconcentrations of rabbit IgG were from 0.22 nM to 132 nM in 1 ml PBSbuffer. The solutions were stirred during all the measurements. FIG. 6Ashow the typical time course of frequency decrease in the presence ofvarious concentrations of rabbit IgG for the MUA/scFv-RG3 sensor. Alinear relationship ranging from 0.22 nM to 33 nM was obtained byplotting the frequency changes versus the concentrations of rabbit IgG(FIG. 6B) for MUA/scFv-RG3 sensor, and 0.33 nM to 33 nM for PSS/scFv-RG3sensors. The frequency change saturated when the concentration of rabbitIgG approached approximately to 132 nM.

A10B scFv-RG3 QCM sensor specificity: The specificity of PSS/scFv-RG3and MUA/scFv-RG3 QCM sensors were examined by adding the followingnegative control reagents (i.e. FBS, goat anti rabbit IgG Fab, antihuman IgG, and yeast) to the detection cells. FIG. 7 shows frequencychange vs. time curves when (a) FBS, (b) goat anti rabbit IgG Fab, (c)anti human IgG, and (d) yeast; curve B: rabbit. IgG were added toPSS/scFv-RG3 (Curve A) and MUA/scFv-RG3 (Curve B) modified Au QCM sensorcells in 1 ml PBS buffer. Shown in FIG. 7, very small nonspecificresponse signals were observed for all selected control reagents. Theseresults indicate that the MUA/scFv-RG3 and PSS/scRV-RG3 modified QCMsensors exhibit excellent antigen specificity.

Comparison of A10B scFv-RG3 sensors to A10B scFv-Cys, scFv-His, andscFv-biotin modified QCM sensors for sensitivity and detection limit:The performances of scFv-RG3 QCM sensors immobilized throughelectrostatic adsorption on negative charged PSS or MUA template werecompared with those early QCM sensors modified by direct adsorption ofscFv-Cys, scFv-RG3, scFv-His on gold surface and adsorption ofscFv-biotin on the pre-coated avidin gold surface. FIG. 8 is acomparison of scFv based QCM sensors' sensitivity. A) MUA/scFv-RG3 B)PSS/scFv-RG3, C) scFv-cys, D) scFv-RG3, E) scFv-His, and F) scFv-biotinon pre-coated surface. All above modified QCM sensors were exposed to132 nM rabbit IgG. These six different modified QCM gold electrodes wereexposed to the 132 nM rabbit IgG solution (FIG. 8). MUA/scFv-RG3modified sensors gave the largest frequency decrease and the lowestdetection limit (0.22 nM). It is eight to ten fold enhancement comparedto scFv-Cys QCM sensor and scFv-His QCM sensor. The PSS/scFv-RG3 gavethe second best sensitivity. These results indicated that monolayernegative charged MUA template allows better oriented immobilization ofscFv RG3 than the anionic polyelectrolyte template. However, both A10BRG3 surface shows superior sensitivity to the earlier engineered A10BscFv-Cys, A10B scFv-His and A10B scFv-biotin indicating the RG3 linkerdesign is effective to be incorporated into scFv for sensor application.

Template effect and A10B scFv Linker sequence effect: Above studiesclearly indicated that scFv-RG3 successfully immobilized onto anionictemplates, and the resulting QCM biosensor exhibited excellentsensitivity and selectivity. To further prove that scFv-RG3 wasimmobilized onto anionic template through electrostatic interaction, twoother templates, a neutral template and a cationic template were testedand compared with MUA SAM. For the neutral template, a long chainalkenthiol, 1-dodecanethiol was used to form SAM. For the cationictemplate, cysteamine, which contained a terminal cationic group, wasused. These three sensors (C₁₂H₂₅SH/scFv-RG3, NH₂CH₂SH/scFv-RG3, andMUA/scFv-RG3) were first treated with scFv-RG3, followed by the additionof the same concentration rabbit IgG. FIG. 9 shows frequency change vs.time curves when MUA/scFv-RG3, C₁₂H₂₅SH/scFv-RG3, and NH₅CS/scFv-RG3 QCMsensors were exposed to the 132 nM rabbit IgG. As shown in FIG. 9,scFv-RG3 through anionic template (MUA/scFv-RG3) gave the largest signalresponse (˜224 Hz). ScFv-RG3 immobilized through neutral template andcationic template showed much weaker adsorption and bound with rabbitIgG poorly (40 Hz and 20 Hz respectively). These studies demonstratedthat the RG3 peptide provides an excellent cationic adsorption sites tobe immobilized on cationic template.

Encouraged by the good sensitivity and specificity of this anionictemplate based QCM sensor, more studies were conducted to understandwhether the multitude positive charged arginine and their positions aswell as the use of spacer glycine are critical for the scFv-RG3 adsorbedon the anionic charged gold surface. Consequently, the scFvs with otherlinkers contained one or two arginines at different locations, scFv-CdS6(Linker: PWIPTPRTFTG, SEQ ID. NO:3) and scFv-ZnS4: (Linker:VISNHAGSSRRL, SEQ ID NO:2), were made. Identical procedures were appliedto immobilize them on MUA or PSS positive charged surface.—FIG. 10 showsfrequency change vs. time curves when different QCM sensors were exposedto the 132 nM rabbit IgG. A) MUA/scFv-RG3, B) PSS/scFv-RG3, C)MUA/scFv-ZnS4, D) PSS/scFv-ZnS4, E) MUA/scFv-CdS6, and F) PSS/scFv-CdS6modified QCM sensors. FIG. 10 shows that the scFv-RG3 gave the largestresponse, scFv-CdS6 produced relative smaller response, and scFv-ZnS4had the smallest response for the addition of 132 nM rabbit IgG.Presumably, the location and the number of arginines played veryimportant roles in the immobilization of the scFvs.

Comparison of different engineered scFvs affinity constant to A10B scFvRG3: The binding between scFv-RG3 and rabbit IgG can be described asEquation 1:

Based on Langmuir adsorption isotherm, association constant (K_(a)) anddissociation constant (K_(d)) for the binding between IgG and scFv-RG3can be evaluated by Equation 2:

$\begin{matrix}{\frac{\left\lbrack {{rabbit}\text{-}{IgG}} \right\rbrack_{0}}{\Delta \; M} = {\frac{\left\lbrack {{rabbit}\text{-}{IgG}} \right\rbrack_{0}}{\Delta \; M_{\max}} + \frac{1}{\Delta \; M_{\max}{Ka}}}} & (2)\end{matrix}$

In this equation, ΔM_(max) is the maximum binding amount, ΔM is themeasured binding amount, and [rabbit-IgG]₀ is the original concentrationof rabbit IgG. For these scFv-RG3 based QCM sensors, plotting[rabbit-IgG]₀/ΔM vs. [rabbit-IgG]₀ by using the data obtained from FIG.6A.—FIG. 11 shows the plot of [rabbit-IgG]₀/ΔM vs. [rabbit-IgG]₀ forMUA/scFv-RG3 modified QCM sensor. As shown in FIG. 11, a linearrelationship was obtained. According to Equation 2, the ratio of theslope to the intercept gave the association constant K_(a) which were9.58×10⁷ M⁻¹ for PSS modified surface, 10.58×10⁷ M⁻¹ for MUA modifiedsurface. K_(d) was calculated as 1/K_(a). Therefore the K_(d) betweenscFv-RG3 and rabbit IgG were 1.04×10⁻⁸ M (PS/RG3), 9.45×10⁻⁹ M(MUA/RG3). Table 2 lists the affinity constants of various engineeredA10B scFvs binding with rabbit IgG antigen. Consistent with earlyresults, the affinity constant for A10B RG3/MUA is highest. Affinityresults for different sensor surfaces were list in Table 2.

Based on the Sauerbrey equation, a surface coverage of 238.3 ng/cm⁻²,corresponding to 8.8×10⁻¹¹ mol/cm⁻² scFv-RG3, was obtained for the MUAmodified QCM surface. The calculated surface coverage and bindingefficiency for different scFv sensors are listed in Table 2.

TABLE 2 Surface coverage and binding efficiency. Surface DetectionLinear coverage limit range QCM sensors (mol/cm²) (nM) (nM) Ka (M⁻¹)MUA/scFv-RG3 8.8 × 10⁻¹¹ 0.2 0.2-33 10.6 × 10⁷  PSS/scFv-RG3 1.8 × 10⁻¹⁰0.33 0.33-33  9.6 × 10⁷ scFv-Cys 1.74 × 10⁻¹⁰  1.7 1.7-66 1.9 × 10⁷scFv-His 6.8 × 10⁻¹¹ 2.3 2.3-33 5.2 × 10⁷

Surface Rigidity: To test the rigidity of these sensor surfaces, QCMimpedance analysis was used to determine the resonator impedance for thebinding events as shown in FIG. 6 through FIG. 9. The change of dampingresistances in all cases was smaller than 1.4% (Table 3).

Changes in damping resistances (|ΔR_(q)|/R_(q)) for experiments shown inFIG. 6 through FIG. 9. FIG. 6a A 0.4% FIG. 6a B 0.7% FIG. 6a C 0.3% FIG.6a D 0.3% FIG. 6a E 0.3% FIG. 6a F 0.8% FIG. 6a G 0.6% FIG. 7 A 0.2%FIG. 7 B 0.1% FIG. 8 A 0.4% FIG. 8 B 0.3% FIG. 8 C 0.5% FIG. 8 D 1.1%FIG. 8 E 1.1% FIG. 8 F 0.2% FIG. 9 A 0.7% FIG. 9 B 0.3% FIG. 9 C 0.6%FIG. 10 A 0.7% FIG. 10 B 0.4% FIG. 10 C 0.8% FIG. 10 D 0.03% FIG. 10 E0.06% FIG. 10 F 0.6%

Conclusions: Single chain antibodies (scFvs) obtained from phage displaylibrary allow rapid isolation of antibodies and their in vitromanipulation at the gene level. They represent smallestimmuno-recognition elements, which provide an emerging strategy in thedevelopment of new immunosensors. Our early work demonstrated that scFvin which either a cysteine (C) amino acid or two histidines isincorporated into the (GGGGS)₃ peptide linker (GGGGS, SEQ ID NO:10) canself-assemble and be immobilized with correct orientation and highsurface concentration on gold. When compared their performance with anIgG monoclonal antibody, the monoclonal IgG Fab and the scFv fragmentsof the IgG monoclonal antibody without cysteine or histidine in thelinker for use in piezoimmunosensors to detect an antigen in a complexbiological serum sample, the scFv-Cys or scFv-His immunosensor displayedgreater assay sensitivity and exhibited less non-specific adsorption.While our recent investigation have shown that by achieving a highdegree of binding site orientation through scFv engineering, withrelatively small size antibody fragments that allows much more densepacking of the binding site, the use of mass detection via QCM fordirect antigen detection becomes a much more realistic analyticaltransduction approach for direct immunosensing. Challenges remain forthe mass production and real world applications of scFvpiezoimmunosensors. Even though literature show that in many scFvs thereseems to be little effect of these linker variations on affinity orstability of the scFv. Certain amino acids in the linker sequence canaffect the yield of functional Fvs that are obtained from refolding ofinclusion bodies. For example, unpaired cysteines, particularlyhydrophobic residues in the linker may reduce the yield of bacterialprotein expression (J. Immun. Methods, 242 (2000)101-114). Thesuccessful implement scFv for immunosensing requires economically massselection and production of scFvs using phage display techniques. Inthis report, we design the peptide linker sequence with cationicarginine charged residue periodicity to favor the adsorption at anioniccharged template surface which facilitates the oriented immobilizationof scFvs on the solid surface. Comparing to early scFv-cys, 10 foldfurther reduction of detection limits and one decade larger of dynamicrange using A10B RG3 were observed. For amino acids, it is the sidechain that gives each amino acid its identity. Protein engineeringallows modifying individual protein molecules so that they are endowedwith self-assembling capability in an oriented manner on the solidsurface. As a result, With 20 amino acids to chose from, our strategypresent a widely applicable technology for engineering scFv forimmunosensing. The described general strategies could avoid thedifficulty of protein expressions when active cysteine or histidine isincorporated and provide a general method to immobilize scFvs in anoriented, ordered or site directed manner on solid surface. We envisionsimilar strategies that incorporate hydrophobic or hydrophilic residueperiodicity to facilitate the adsorption on a non-polar or polarsurface.

EXAMPLE 2

This example is a surface plasmon resonance (SPR) study of2-mercaptoethaol as a template in A10B scFv-YG immobilization.

Motivation: We have created an innovative immobilization method that iseasy to operate, efficient, less costing and reliable in constructingbiosensors for non-labeled immunoassay for understanding antibody andantigen interaction. Traditional immunoassays like ELISA are veryreliable but is also a time- and cost-consuming process. For example,two antibodies that are specific for different sites on the same antigenare needed. It is not always easy to find such proper antibody pair forthe assay. On the contrary, non-labeled immunosensors will do assays ina simple one step fashion. The sensing element, most likely theantibody, is immobilized on the surface of a transducer. When theantibody interacts with the antigen, mechanical or optical properties ofthe transducer are also changed. Such change is recorded and analyzed toprovide information of the antibody-antigen interaction.

Background: The key component of antibody biosensors or immunosensors isthe immobilized antibody present on the transducer surface.Immunosensors have been reported for detection and analysis of thepathogenic proteins, bacteria, and viruses. In order to improve theperformance and sensitivity of immunosensors, our group has developed atechnique which takes advantage of engineered recombinant single-chainfragment variable (scFv) antibody. Biosensors based on this techniqueare named scFv biosensors.

The scFv is the smallest portion of an antibody that retains thespecificity and the function of recognition of antigen as illustrated inFIG. 12. It comprises the antibody heavy-chain (V_(H)) and light-chain(V_(L)) variable domains that are connected by a peptide linker tostabilize the molecule (FIG. 12). Compared to a whole antibody, the scFvis much smaller in size, making it possible to be immobilized ontransducer surface with high density, which greatly increases thesensitivity. High density of sensing elements also allows us to performa non-regeneration kinetic assay using non-labeled surface plasmonresonance (SPR) technique we developed.

One challenge in constructing scFv biosensors is the orientation of theimmobilized scFv. Undesired orientation may deteriorate the performanceof scFv biosensors. In this example, we develop immobilization methodsthat take advantage of the limitless flexibility of antibody engineeringfor the scFvs with the inherent quick, clean, high fidelity charactersof surface coupling chemistry (e.g. electrostatic, hydrogen bonding orcovalent attachment) to attach scFvs to a pre-formed functionalizedself-assembled monolayer (SAM) template. Shown in FIG. 13, the templatewill help scFv to be immobilized with desired orientation, i.e. amonolayer of chemical molecules on the template will interact with thescFv linker so that the scFv is immobilized upright; therefore, thefunctional tip of scFv is pointing outwards in the solution phasewithout steric hindrance.

Innovation and Experiment: The selection of template is based on thesequence of the linker. The linker is a peptide containing 10-20 aminoacids. In a YG linker, tyrosine is a potential hydrogen bond formationsite because of the phenol group in tyrosine. Therefore, we propose touse 2-mercaptoethanol (HS—CH₂—CH₂—OH) as the template. The thiol group(HS—) of 2-mercaptoethanol helps the molecule to form a stable monolayeron transducer surface which is gold. The hydroxyl group (—OH) at theother end of 2-mercaptoethanol can form a hydrogen bond with the phenolgroup in the scFv linker. This bonding is strong enough to hold the scFvon the transducer surface as well as to keep the scFv in the desiredorientation. To test this innovative idea, we will compare theperformance of scFv immobilized through 2-mercaptoethanol to theperformance of scFv immobilized directly on the transducer surface.

In this example, A10B anti-rabbit IgG scFv is used. Biosensors areconstructed by means of direct immobilization or immobilization through2-mercaptoethanol. The immobilization template is a piece of glasscoated with gold, which is suited for SPR analysis.

Our innovative work on non-labeled antibody immunoassay shows manyadvantages over traditional immunoassay such as ELISA. In order for mosttraditional immunoassays to work, the assays need two antibodies thatare specific for different sites on the same antigen. It can beextremely difficult and time-consuming to find pairs of antibodies thatwork in these assays even if there are a lot of antibodies to work with.The advantage of our non-labeled assay system is that only one antibodyis needed to detect an antigen as well as the high sensitivity andspecificity comparing to those immunosensors current available.

Experimental results: In the following experiment, a template,2-mercaptoethanol, enhances the performance of A10B-YG scFv/A10B-FP1scFv/A10B-ZnS4 scFv in recognizing rabbit IgG (r-IgG). As one amino acidin the YG/FP1/ZnS4 linker contains a phenol group and it can formhydrogen bonds with the hydroxyl group in 2-mercatoethanol, we expectthe presence of the template can increase the amount and/or improveorientation of A10B-YG/FP1/ZnS4 scFv. Electrochemical impedancespectroscopy is used to verify the findings obtained by surface plasmonresonance (SPR).

Linker Peptide Sequences: The A10B scFv YG linker amino acid sequence isYGGYG(GGGS)₂, ie. YGGYGGGGSGGGS (SEQ ID NO:11) The A10B scFv FP1 linkeramino acid sequence is SVSVGMKPSPRP (SEQ ID NO:4). The A10B scFvZnS4linker amino acid sequence is VISNHAGSSRRL (SEQ ID NO:2).

Experimental.

All the SPR sensorgrams in the following part show the differencebetween two parallel sensors on two flow cells, i.e. FC1 and FC2. FC1 isused as reference cell, FC2 is used as sample cell.

The design of this experiment was to compare the template-immobilizedscFv to the directly immobilized scFv side by side. Block reagent, suchas BSA, was not used. The reference flow cell (FC1) was directlyimmobilized with A10B-YG scFv, while the sample flow cell (FC2) wasimmobilized with the same scFv through the template.

FIG. 19 illustrates the SPR response difference between A10B scFv-Cysbiosensor reference cell (FC1) and sample cell (FC2). The graph containsseven injection steps that correspond to seven different concentrationsof rabbit IgG, 6.25, 12.5, 25, 50, 100, and twice 200 μg/mL or 0.042,0.083, 0.17, 0.33, 0.67, and twice 1.3 μM in that order. In thisexperiment, FC1 has the injection order of A10B-YG, R-IgG. FC2 has theinjection order of 2-mercaptoethanol, A10B-YG, R-IgG.

The same amount of R-IgG generated larger response on the flow cell withtemplate indicating that the hydrogen bonding template allows betterimmobilization of scFvs. The response difference was about 700 RU.

FIG. 20 illustrates the ELISA results of sensitivity and specificity ofA10B YG and A10B RG3. FIG. 20 shows the ELISA results of comparison ofthe sensitivity and specificity of A10B YG and RG3 when they react withrabbit IgG and BSA. Both A10B YG and RG3 has similar level ofnon-specific binding with BSA. But A10B YG has smaller sensitivity thanA10B RG3. Amounts in descending order from left to right as listed inthe key.

Chip regeneration: FIG. 21 shows that 30 μg/mL 0.3 M Zwittergent 3-08 pH2.0 was used to regenerate the sensor surface. Then it was reused todetect rabbit IgG. Some loss of the sensitivity was observed but thesensor could still gives reasonable response after the regeneration.When the regeneration process is optimized, we expect that the minimizedsensitivity loss will be observed so that the sensor can have long lifetime. In this experiment, (1) FC1 has the injection order of A10B-YG,R-IgG. FC2 has the injection order of 2-mercaptoethanol, A10B-YG, R-IgG.(2)30 μg/mL 0.3 M Zwittergent 3-08 pH 2.0. (3) repeat the experiment of(1)

Discussion.

The direct immobilization of A10B-YG scFv on gold plate did not giveacceptable result (Experiment 3) because the poorly orientated scFvlimited the bioactivity of scFv.

In order to improve the performance of A10B-YG scFv, we proposed scFvimmobilization through a template, 2-mercaptoethanol, utilizing theformation of hydrogen bonding between atoms O—H—O. The template improvedthe performance of A10B-YG scFv and retained its sensitivity andspecificity.

Although the performance of A10B-YG can be improved with the help of atemplate, it still can not compare to the performance of A10B-cys. TheA10B-YG sensor is saturated when the concentration of r-IgG reached 50ug/mL while A10B-cys can go as high as 200 ug/mL. This could be due tothe original low sensitivity of A10B YG comparing to A10B scFv-cys andA10B RG3, not due to the immobilization protocol.

Conclusion.

The idea has been verified that a poorly performed scFv can be improvedby using template in immobilization. The most possible reason for2-mercaptoethanol improving the immobilization of A10B-YG is due to thehydrogen bonding between atoms O—H—O or N—H—O.

EXAMPLE 3

This example illustrates how scFv QCM can be used to detect bacteria. Wehave demonstrated that the scFv biosensor system can be used to providea platform immobilization strategy to build rigid IgG Fc receptorlayers. Just as important, the ability of immunoglobulins to react withother molecules at sites located outside the antigen-combining site,related to the effector functions of antibodies, are the most importantpart of the immune response including from such well-known reactions asthe activation of the complement cascade and the activation or theinhibition of cells after binding with the Fc receptors totransportation of immunoglobulins through cell membranes. Shown in FIG.1, monomeric scFv allows uniform 2:1 binding with rabbit IgG CH1 region,this results a highly oriented IgG Fc portion pointing toward solutionphase for the detection of Fc receptors. This feasibility was proved bydetection of Protein A, an Fc receptor on the cell wall ofStaphylococcus aureus bacteria. Detection of protein A in bacteria ischallenging due to the presence of excess antibody in the bacteriaculture that often seriously suppresses the response of an assay bycompeting for the Fc binding sites on protein A thereby reducing thesensitivity of the assay. We demonstrated that the specific orientationof Fc capture agents consistently increases the analyte-binding capacityof the surfaces, with up to 7-fold improvements over surfaces withrandomly oriented capture agents. Randomly attached IgG could not bepacked at such a high density and had a lower specific activity. Theseresults emphasize the importance of immobilization of capture reagentsto surfaces such that their binding sites are oriented toward thesolution phase.

The methods we demonstrated in this report can be quite general forbuilding up rigid films for acoustic detection of various reagents fromproteins to cell or bacterial. In contrast to SPR technique which probesthe changes in the effective refractive index (RI) of the guided wavescaused by the interactions of their evanescent field with analytemolecules binding specifically to their reaction partners immobilized onthe sensor surfaces and has a limit of the thickness of the biofilms,i.e. less than 200 nm, QCM sensor detects only those materials that areacoustically coupled to the sensor surface and theoretically it has nolimits about the thickness of the film. But it only requires theimmobilized biofilms with high rigidity so it can acoustically couplewith quartz oscillation. Since the peptide chain length, amino acidcomposition, specific sequence, net charge at neutral pH andhydrophobicity, all these features could be controlled and have dramaticinfluence on the performance of scFv based QCM sensors, For example,15-mer unmodified peptides, there are (˜10²⁴) diverse sequences byconsidering the 20 common amino acids alone, Thus, the linker peptidedesign provides huge possibilities for surface coupling methodology.This feature, in conjunction with the real-time, non-labeled charactersof QCM, presents a widely applicable protein immobilization technologyfor investigating the protein-protein interactions in general andpromises QCM to be an indispensable technique in mapping theligand-receptor interactions affordable to most investigators in lifescience research.

Chemicals and Materials.

Bacterial Staphylococcus aureus cultures and sample preparation: Thebacterial Staphylococcus aureus, Cowan's serotype 1 that specificproduce protein A was purchased from ATCC (Cat. number 12598, ATCC,Manassas, Va., USA). Bacteria was inoculated into 100 ml of NutrientBroth culture medium contains 3.0 gm Beef Extract and 5.0 gm Peptone inone liter water, autoclave at 121° C. for 15 minutes. (Difco Cat. #233000, BD, Sparks, Md., USA). Bacteria were incubated at 37° C. forovernight with shaking at 150 rpm. The bacterial was then centrifugedown at 4,000 rpm. Supernatant was discarded. The bacteria were washedthree times by re-suspended pellet in PBS and centrifuge at same speedas above. The washed Staphylococcus aureus bacteria were collected intomicrocentrifuge tubes for assay. To remove bacteria surface bound IgG,bacteria sample was well re-suspended in 0.1 M Na-citric acid elutionbuffer (pH 2.8) for 10 minutes, washed three times by PBS to neutralizepH to 7.0 and centrifuge at 4,000 rpm. The cell numbers were counted byusing a hemacytometer counting chamber.

A10B ScFv immobilization: The non-polished gold quartz surface wascleaned sequentially with mixed concentrated acid solution (H₂SO₄/HNO₃in 1:1 v/v), biograde water, and ethanol three times. Then, the surfacewas rinsed with biograde water and dried with nitrogen. The freshlywashed gold surface was first immersed either into 4 mM MUA solutionovernight or in 2 mg/ml PSS solution for 1-2 hours to form self-assemblyanionic charged layer. The anionic charged gold surface was thenimmersed into A10B scFv-RG3 solution for 8 hours, followed by thetreatment of blocking reagent, 0.1% BSA, for 0.5 hour. After theincubation, the excess scFvs on the surface of QCM was washed away withPBS buffer and biograde water. The MUA/scFv-RG3 or PSS/scFv-RG3 modifiedsurface was dried under nitrogen, and used for the detection. A similarprocedure was applied to form the PSS/scFv-RG3 modified surface.

QCM measurements: The gold quartz crystal electrode used through outthis study was AT cut quartz that coated with 1000 Å gold in ˜0.23 cm²geometric area (International Crystal Company, Oklahoma). It was cleanedas above and modified with scFvs. Then it was mounted in a Kel-F cellsealed by two O-rings and filled with 1 mL PBS buffer. The cell wascontinuously stirred during the measurement and was placed in a Faradaycage to reduce the potential electromagnetical noise. The frequencychange and the damping resistance change caused by the analyte additionwere monitored by the Network/Spectrum/Impedance Analyzer (Agilent4395A).

Results and Discussion.

QCM analysis of purified protein A: The orientated immobilization ofscFv-RG3 on the MUA surface specifically bound with the CH1 domain ofthe rabbit IgG to form what is referred to as an MUA/scFv-RG3/IgGsurface. This made the IgG Fc portion available in a highly packedmanner. The highly oriented IgG Fc layer could be applied for thedetection of Fc receptors. Protein A, a well-known Fc receptor, is ableto specifically bind to the Fc portion of various classes and subclassesof immunoglobins, and especially to IgG. Thus, we selected purifiedprotein A as a model sample to examine the detection ability of an Fcreceptor via our immobilization strategy.

Addition of purified protein A sample to the MUA/scFv-RG3/IgG surface(ie. the rabbit IgG was already bound to the MUA/scFv-RG3 modifiedsurface) produced a 47 Hz signal response. This signal is about 7-foldincrease over randomly orientated rabbit IgG surface and protein A/IgGsurface (rabbit IgG was coupled onto the protein A surface) (FIG. 14).To investigate the specificity of the protein A detection, theMUA/scFv-RG3 sensor surface in the absence of rabbit IgG was used asnegative control. The addition of protein A to the control surface onlygenerated a very small frequency decrease (FIG. 14 Curve d). FIG. 14illustrates frequency change vs. time curves when protein A was added to(a) MUA/scFv-RG3 coupling with rabbit IgG surface (MUA/scFv-RG3/IgGsurface); (b) randomly orientated rabbit IgG surface; (c) rabbit IgGcoupling onto the protein A surface; (d) MUA/scFv-RG3 surface withoutrabbit IgG (negative control).

QCM analysis of Staphylococcus aureus: In order to examine the sensorselectivity, E. coli was utilized as a negative control. The addition ofE. coli to MUA/scFv-RG3/IgG sensor gave a really small signal response.After the exposure to E. coli, Staphylococcus aureus, gram-positivebacteria that possess protein A on cell surface, were consecutivelyadded to the sensor. (The Staphylococcus aureus sample was pretreated by0.1 M Na-citric acid elution buffer (pH 2.8) to remove the IgG bond onthe surface). FIG. 15 shows frequency vs. time curves. MUA/scFv-RG3/IgGsurface was exposed to 2.6×10⁸ cells/ml E. coli (negative control,),followed by the addition of Staphylococcus aureus (2.5×10⁸ cells/ml,1.2×10⁹ cells/ml). Large response signals were observed upon theaddition of Staphylococcus aureus sample (FIG. 15). This resultindicated that the sensor has good selectivity toward Staphylococcusaureus, and the sensor can remain the high sensitivity towardStaphylococcus aureus even in a complex solution system.

Binding Study: FIG. 16 shows the frequency vs, time curve when differentconcentrations of Staphylococcus aureus samples were added toMUA/scFv-RG3 coupling with rabbit IgG surface.

Selectivity Test: FIG. 17 shows the frequency vs, time curve whenMUA/scFv-RG3 coupling with rabbit IgG surface was exposed to 1.5×10⁸cells/ml E. coli (negative control) and 9.8×10⁸ cells/ml Staphylococcusaurenus.

In order to test the sensor specificity, several control experimentswere then performed. (FIG. 18). Different samples were added to theMUA/scFv-RG3/IgG sensors. The addition of Staphylococcus aureus sample(1.4×10⁹ cell/ml) that was pre-treated with acid to remove all surfaceIgG to generate a large frequency change (˜230-250 Hz, Bar A).Staphylococcus aureus without acid treatment (1.4×10⁹ cell/ml) gave only˜44 Hz frequency change (Bar B). This ˜6 times signal reductionsuggested that some protein A on the Staphylococcus aureus surface havebeen occupied already by the surface bond IgG. Thus, removal of the IgGfrom the bacterial surface can dramatically increase the sensitivity ofthe sensor. E. coli, a gram-negative bacterium, was applied as negativecontrol reagent, and generated really small frequency change (5 Hz, BarC).

The directly immobilized rabbit IgG surface (Bar D), MUA/scFv-RG3surface (without the treatment of rabbit IgG) (Bar E), and mannosemodified QCM surfaces (specifically bind to Con A) (Bar F) were selectedas control surfaces. The addition of the same concentration of acidtreated Staphylococcus aureus generated ˜39 Hz frequency change to therandom orientated rabbit IgG surface (Bar D), ˜10 Hz non-specificabsorption to the MUA/scFv-RG3 surface (without rabbit IgG) (Bar E) andno detectable nonspecific absorption to the mannose modified QCMsurfaces (Bar F).

All these control experiments confirmed that the MUA/scFv-RG3/IgGsurface is highly selective for the detection Staphylococcus aureus viathe specific binding between Fc portion of rabbit IgG and cell surfaceprotein A.

While the present invention is described herein with reference toillustrated embodiments, it should be understood that the invention isnot limited hereto. Those having ordinary skill in the art and access tothe teachings herein will recognize additional modifications andembodiments within the scope thereof. Therefore, the present inventionis limited only by the Claims attached herein.

1. An apparatus comprising: (a) a substrate with an exposed surface; (b)a compound provided as a layer, bound to the solid substrate; and (c) aplurality of recombinant single chain antibodies (scFv's) specific forthe target molecule and bound to the compound on the solid substrate,each scFv comprising an antibody variable light chain (V_(L))polypeptide specific for the target molecule, an antibody variable heavychain (V_(H)) polypeptide specific for the target molecule, and a linkerpolypeptide covalently linking the antibody variable light chain (VL)polypeptide to the antibody variable heavy chain (VH) polypeptide, thelinker polypeptide having an amino acid sequence comprising one or moreamino acids with sidechains that bind to the compound, wherein the scFvis capable of binding the target molecule to the solid substrate, whenprovided to the apparatus.
 2. The apparatus of claim 1, wherein therecombinant single chain antibodies (scFv's) are specific forimmunoglobulins as the target molecules, further comprising a pluralityof the immunoglobulins bound to the scFv molecules so that Fc regions ofthe immunoglobulins are exposed as a binding layer for Fc receptors. 3.The apparatus of claim 1, wherein the target molecules are analytes ofinterest in a sample and the apparatus detects whether the analyte ofinterest is present in the sample.
 4. The apparatus of claim 1, whereinthe compound and the amino acid sidechains form electrostaticinteractions.
 5. The apparatus of claim 1, wherein the compound and theamino acid sidechains form hydrogen bonds.
 6. The apparatus of claim 1,wherein the amino acids with sidechains are arginine or tyrosine.
 7. Theapparatus of claim 1, wherein the compound is electrostatically charged.8. The apparatus of claim 1, wherein the compound is an anionicpolyelectrolyte or 2-mercaptoethanol.
 9. The apparatus of claim 8,wherein the anionic polyelectrolyte is poly(sodium 4-styrenesulfonate)(PSS) or 11-mercaptoundecanoic acid (MUA).
 10. The apparatus of claim 1,wherein the substrate is gold.
 11. The apparatus of claim 1, wherein theapparatus is provided as a binding component of an immunosensor.
 12. Theapparatus of claim 11, wherein the immunosensor is a quartz crystalmicrobalance (QCM) device or a surface plasmon resonance (SPR) device.13. The apparatus of claim 1, wherein the apparatus is provided as amicrotiter plate for an ELISA assay or as an affinity matrix forimmunopurification.
 14. The apparatus of claim 1, wherein the aminoacids with sidechains are separated by one or more spacer amino acids.15. The apparatus of claim 14, wherein the spacer amino acids areselected from the group consisting of glycine and serine.
 16. Theapparatus of claim 1, wherein the amino acid sequence of the linkerpolypeptide comprises a series of two or more arginine-glycine (RG)repeats.
 17. The apparatus of claim 1, wherein the amino acid sequenceof the linker polypeptide comprises a sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:11, and SEQ ID NO:12.
 18. A method of detecting ananalyte of interest in a sample comprising: (a) providing the sample;(b) providing an immunosensor device having a component apparatus forbinding the analyte of interest in a sample comprising: a substrate withan exposed surface; a compound provided as a layer bound on the solidsubstrate; and a plurality of recombinant single chain antibodies(scFv's) specific for the analyte of interest bound to the compound onthe solid substrate each scFv comprising an antibody variable lightchain (V_(L)) polypeptide specific for an analyte, an antibody variableheavy chain (V_(H)) polypeptide specific for the analyte, and a linkerpolypeptide covalently linking the antibody variable light chain (VL)polypeptide to the antibody variable heavy chain (VH) polypeptide, thelinker polypeptide having an amino acid sequence comprising one or moreamino acids with sidechains that bind to the compound, wherein when theanalyte of interest is present in the sample the scFv binds the analyteto the solid substrate; (c) applying the sample to the apparatus for atime sufficient for the scFv on the substrate to bind to the analyte ifpresent in the sample; and (d) detecting the analyte bound to the scFvon the solid substrate with the immunosensor device.
 19. The method ofclaim 18, wherein the immunosensor device is a quartz crystalmicrobalance (QCM) device or a surface plasmon resonance (SPR) device.20. A method of binding an analyte of interest in a sample comprising:(a) providing the sample; (b) providing an apparatus for binding ananalyte of interest in a sample comprising: a substrate with an exposedsurface; a compound provided as a layer bound on the solid substrate;and a plurality of recombinant single chain antibodies (scFv's) specificfor the analyte of interest bound to the compound on the solid substrateeach scFv comprising an antibody variable light chain (V_(L))polypeptide specific for an analyte, an antibody variable heavy chain(V_(H)) polypeptide specific for the analyte, and a linker polypeptidecovalently linking the antibody variable light chain (VL) polypeptide tothe antibody variable heavy chain (VH) polypeptide, the linkerpolypeptide having an amino acid sequence comprising one or more aminoacids with sidechains that bind to the compound, wherein when theanalyte of interest is present in the sample the scFv binds the analyteto the solid substrate; and (c) incubating the sample to the apparatusfor a time sufficient for the scFv on the solid substrate to bind to theanalyte if present in the sample.
 21. The method of claim 20, whereinthe apparatus is a microtiter plate for an ELISA assay or a microarray.22. The method of claim 20, wherein the apparatus is an affinity column.23. A method of determining whether an analyte with an Fc receptor ispresent in a sample comprising: (a) providing the sample; (b) providingan immunosensor device having a component an apparatus comprising: asubstrate with an exposed surface; a compound provided as a layer, boundto the solid substrate; a plurality of recombinant single chainantibodies (scFv's) specific for an immunoglobulin and bound to thecompound on the solid substrate, each scFv comprising an antibodyvariable light chain (V_(L)) polypeptide specific for theimmunoglobulin, an antibody variable heavy chain (V_(H)) polypeptidespecific for the immunoglobulin, and a linker polypeptide covalentlylinking the antibody variable light chain (VL) polypeptide to theantibody variable heavy chain (VH) polypeptide, the linker polypeptidehaving an amino acid sequence comprising one or more amino acids withsidechains that bind to the compound, wherein the scFv is capable ofbinding the immunoglobulin to the solid substrate; and a plurality ofthe immunoglobulins bound to the scFv molecules so that Fc regions ofthe immunoglobulins are exposed as a binding layer for the Fc receptor;(c) applying the sample to the apparatus for a time sufficient for thescFv on the substrate to bind to the Fc receptor if present in thesample; (d) detecting the Fc receptors in the sample bound to the scFvon the solid substrate with the immunosensor device; and (e) determiningwhether the analyte is present in the sample by the result in step (d).24. The method of claim 23, wherein the immunosensor device is a quartzcrystal microbalance (QCM) device or a surface plasmon resonance (SPR)device.
 25. The method of claim 23, wherein the Fc receptor is Protein Aor Protein G.
 26. The method of claim 23, wherein the analyte with theFc receptor is Staphylococcus aureus.
 27. The method of claim 23,wherein the immunoglobulin is IgG.